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The shift to a genotype-first approach in genetic diagnostics has revolutionized our understanding of neurodevelopmental disorders, expanding both their molecular and phenotypic spectra. Kleefstra syndrome (KLEFS1) is caused by EHMT1 haploinsufficiency and exhibits broad clinical manifestations. EHMT1 encodes euchromatic histone methyltransferase-1-a pivotal component of the epigenetic machinery. We have recruited 209 individuals with a rare EHMT1 variant and performed comprehensive molecular in silico and in vitro testing alongside DNA methylation (DNAm) signature analysis for the identified variants. We (re)classified the variants as likely pathogenic/pathogenic (molecularly confirming Kleefstra syndrome) in 191 individuals. We provide an updated and broader clinical and molecular spectrum of Kleefstra syndrome, including individuals with normal intelligence and familial occurrence. Analysis of the EHMT1 variants reveals a broad range of molecular effects and their associated phenotypes, including distinct genotype-phenotype associations. Notably, we showed that disruption of the "reader" function of the ankyrin repeat domain by a protein altering variant (PAV) results in a KLEFS1-specific DNAm signature and milder phenotype, while disruption of only "writer" methyltransferase activity of the SET domain does not result in KLEFS1 DNAm signature or typical KLEFS1 phenotype. Similarly, N-terminal truncating variants result in a mild phenotype without the DNAm signature. We demonstrate how comprehensive variant analysis can provide insights into pathogenesis of the disorder and DNAm signature. In summary, this study presents a comprehensive overview of KLEFS1 and EHMT1, revealing its broader spectrum and deepening our understanding of its molecular mechanisms, thereby informing accurate variant interpretation, counseling, and clinical management., Competing Interests: Declaration of interests A.M.M. is an employee of GeneDx, LLC. B.S. is a shareholder in EpiSign Inc., a biotechnology company involved in commercialization of EpiSign technology., (Copyright © 2024 American Society of Human Genetics. All rights reserved.)

Rare or novel missense variants in large genes such as TTN and NEB are frequent in the general population, which hampers the interpretation of putative disease-causing biallelic variants in patients with sporadic neuromuscular disorders. Often, when the first initial genetic analysis is performed, the reconstructed haplotype, i.e. phasing information of the variants is missing. Segregation analysis increases the diagnostic turnaround time and is not always possible if samples from family members are lacking. To overcome this difficulty, we investigated how well the linked-read technology succeeded to phase variants in these large genes, and whether it improved the identification of structural variants. Linked-read sequencing data of nemaline myopathy, distal myopathy, and proximal myopathy patients were analyzed for phasing, single nucleotide variants, and structural variants. Variant phasing was successful in the large muscle genes studied. The longest continuous phase blocks were gained using high-quality DNA samples with long DNA fragments. Homozygosity increased the number of phase blocks, especially in exome sequencing samples lacking intronic variation. In our cohort, linked-read sequencing added more information about the structural variation but did not lead to a molecular genetic diagnosis. The linked-read technology can support the clinical diagnosis of neuromuscular and other genetic disorders., (© 2024. The Author(s).)

Syndromic constitutive thrombocytopenia encompasses a heterogeneous group of disorders characterised by quantitative and qualitative defects of platelets while featuring other malformations. Recently, heterozygous, de novo variants in RAP1B were reported in three cases of syndromic thrombocytopenia. Here, we report two additional, unrelated individuals identified retrospectively in our data repository with heterozygous variants in RAP1B: NM_001010942.2(RAP1B):c.35G>A, p.(Gly12Glu) (de novo) and NM_001010942.2(RAP1B):c.178G>A, p.(Gly60Arg). Both individuals had thrombocytopenia, as well as congenital malformations, and neurological, behavioural, and dysmorphic features, in line with previous reports. Our data supports the causal role of monoallelic RAP1B variants that disrupt RAP1B GTPase activity in syndromic congenital thrombocytopenia., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The multiple pterygium syndromes (MPS) are rare disorders with disease severity ranging from lethal to milder forms. The nonlethal Escobar variant MPS (EVMPS) is characterized by multiple pterygia and arthrogryposis, as well as various additional features including congenital anomalies. The genetic etiology of EVMPS is heterogeneous and the diagnosis has been based either on the detection of pathogenic CHRNG variants (~23% of patients), or suggestive clinical features. We describe four patients with a clinical suspicion of EVMPS who manifested with multiple pterygia, mild flexion contractures of several joints, and vertebral anomalies. We revealed recessively inherited MYH3 variants as the underlying cause in all patients: two novel variants, c.1053C>G, p.(Tyr351Ter) and c.3102+5G>C, as compound heterozygous with the hypomorphic MYH3 variant c.-9+1G>A. Recessive MYH3 variants have been previously associated with spondylocarpotarsal synostosis syndrome. Our findings now highlight multiple pterygia as an important feature in patients with recessive MYH3 variants. Based on all patients with recessive MYH3 variants reported up to date, we consider that this disease entity should be designated as "Contractures, pterygia, and variable skeletal fusions syndrome 1B," as recently suggested by OMIM. Our findings underline the importance of analyzing MYH3 in the differential diagnosis of EVMPS, particularly as the hypomorphic MYH3 variant might remain undetected by routine exome sequencing., (© 2020 Wiley Periodicals LLC.)

Fetal akinesia deformation sequence (FADS) and lethal multiple pterygium syndrome (LMPS) are clinically overlapping syndromes manifesting with reduced or absent fetal movement, arthrogryposis, and several anomalies during fetal life. The etiology of these syndromes is heterogeneous, and in many cases it remains unknown. In order to determine the genetic etiology of FADS in two fetuses with fetal akinesia, arthrogryposis, edema, and partial cleft palate, we utilized exome sequencing. Our investigations revealed a homozygous nonsense variant [c.1116C>A, p.(Cys372Ter)] in the SLC18A3 gene, which encodes for the vesicular acetylcholine transporter (VAChT) responsible for active transport of acetylcholine in the neuromuscular junction. This is the first description of a nonsense variant in the SLC18A3 gene, as only missense variants and whole gene deletions have been previously identified in patients. The previously detected SLC18A3 defects have been associated with congenital myasthenic syndromes, and therefore our findings extend the clinical spectrum of SLC18A3 defects to severe prenatal phenotypes. Our findings suggest that nonsense variants in SLC18A3 cause a more severe phenotype than missense variants and are in line with previous studies showing a lethal phenotype in VAChT knockout mice. Our results underline the importance of including SLC18A3 sequencing in the differential diagnostics of fetuses with arthrogryposis, FADS, or LMPS of unknown etiology., (© 2019 Wiley Periodicals, Inc.)

We used a comprehensive metabolomics approach to study the altered urinary metabolome of two mitochondrial myopathy, encephalopathy lactic acidosis and stroke like episodes (MELAS) cohorts carrying the m.3243A>G mutation. The first cohort were used in an exploratory phase, identifying 36 metabolites that were significantly perturbed by the disease. During the second phase, the 36 selected metabolites were able to separate a validation cohort of MELAS patients completely from their respective control group, suggesting usefulness of these 36 markers as a diagnostic set. Many of the 36 perturbed metabolites could be linked to an altered redox state, fatty acid catabolism and one-carbon metabolism. However, our evidence indicates that, of all the metabolic perturbations caused by MELAS, stalled fatty acid oxidation prevailed as being particularly disturbed. The strength of our study was the utilization of five different analytical platforms to generate the robust metabolomics data reported here. We show that urine may be a useful source for disease-specific metabolomics data, linking, amongst others, altered one-carbon metabolism to MELAS. The results reported here are important in our understanding of MELAS and might lead to better treatment options for the disease., (Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitochondrial disorders (MDs) are inherited multi-organ diseases with variable phenotypes. Inclusion body myositis (IBM), a sporadic inflammatory muscle disease, also shows mitochondrial dysfunction. We investigated whether primary and secondary MDs modify metabolism to reveal pathogenic pathways and biomarkers. We investigated metabolomes of 25 mitochondrial myopathy or ataxias patients, 16 unaffected carriers, six IBM and 15 non-mitochondrial neuromuscular disease (NMD) patients and 30 matched controls. MD and IBM metabolomes clustered separately from controls and NMDs. MDs and IBM showed transsulfuration pathway changes; creatine and niacinamide depletion marked NMDs, IBM and infantile-onset spinocerebellar ataxia (IOSCA). Low blood and muscle arginine was specific for patients with m.3243A>G mutation. A four-metabolite blood multi-biomarker (sorbitol, alanine, myoinositol, cystathionine) distinguished primary MDs from others (76% sensitivity, 95% specificity). Our omics approach identified pathways currently used to treat NMDs and mitochondrial stroke-like episodes and proposes nicotinamide riboside in MDs and IBM, and creatine in IOSCA and IBM as novel treatment targets. The disease-specific metabolic fingerprints are valuable "multi-biomarkers" for diagnosis and promising tools for follow-up of disease progression and treatment effect., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

We studied the genetic background of a family with SCA, showing dominant inheritance and anticipation. Muscle histology, POLG1 gene sequence, neuropathology and mitochondrial DNA analyses in a mother and a son showed typical findings for a mitochondrial disorder, and both were shown to be homozygous for a recessive POLG1 mutation, underlying mitochondrial recessive ataxia syndrome, MIRAS. The healthy father was a heterozygous carrier for the same mutation. Recessively inherited MIRAS mutations should be tested in dominantly inherited SCAs cases of unknown cause, as the high carrier frequency of MIRAS may result in two independent introductions of the mutant allele in the family and thereby mimic dominant inheritance., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Muscle biopsy is the gold standard for diagnosis of mitochondrial disorders because of the lack of sensitive biomarkers in serum. Fibroblast growth factor 21 (FGF-21) is a growth factor with regulatory roles in lipid metabolism and the starvation response, and concentrations are raised in skeletal muscle and serum in mice with mitochondrial respiratory chain deficiencies. We investigated in a retrospective diagnostic study whether FGF-21 could be a biomarker for human mitochondrial disorders., Methods: We assessed samples from adults and children with mitochondrial disorders or non-mitochondrial neurological disorders (disease controls) from seven study centres in Europe and the USA, and recruited healthy volunteers (healthy controls), matched for age where possible, from the same centres. We used ELISA to measure FGF-21 concentrations in serum or plasma samples (abnormal values were defined as >200 pg/mL). We compared these concentrations with values for lactate, pyruvate, lactate-to-pyruvate ratio, and creatine kinase in serum or plasma and calculated sensitivity, specificity, and positive and negative predictive values for all biomarkers., Findings: We analysed serum or plasma from 67 patients (41 adults and 26 children) with mitochondrial disorders, 34 disease controls (22 adults and 12 children), and 74 healthy controls. Mean FGF-21 concentrations in serum were 820 (SD 1151) pg/mL in adult and 1983 (1550) pg/mL in child patients with respiratory chain deficiencies and 76 (58) pg/mL in healthy controls. FGF-21 concentrations were high in patients with mitochondrial disorders affecting skeletal muscle but not in disease controls, including those with dystrophies. In patients with abnormal FGF-21 concentrations in serum, the odds ratio of having a muscle-manifesting mitochondrial disease was 132·0 (95% CI 38·7-450·3). For the identification of muscle-manifesting mitochondrial disease, the sensitivity was 92·3% (95% CI 81·5-97·9%) and specificity was 91·7% (84·8-96·1%). The positive and negative predictive values for FGF-21 were 84·2% (95% CI 72·1-92·5%) and 96·1 (90·4-98·9%). The accuracy of FGF-21 to correctly identify muscle-manifesting respiratory chain disorders was better than that for all conventional biomarkers. The area under the receiver-operating-characteristic curve for FGF-21 was 0·95; by comparison, the values for other biomarkers were 0·83 lactate (p=0·037, 0·83 for pyruvate (p=0·015), 0·72 for the lactate-to-pyruvate ratio (p=0·0002), and 0·77 for creatine kinase (p=0·013)., Interpretation: Measurement of FGF-21 concentrations in serum identified primary muscle-manifesting respiratory chain deficiencies in adults and children and might be feasible as a first-line diagnostic test for these disorders to reduce the need for muscle biopsy., Funding: Sigrid Jusélius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppö Foundation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The clinical phenotypes vary considerably and can be divided into three groups: 1) childhood-onset encephalopathy and hepatopathy, 2) juvenile onset refractory epilepsy and migraine-like headaches, and 3) adult-onset ataxia and neuropathy with additional symptoms such as psychiatric symptoms and cognitive impairment. The life-threatening MIRAS epilepsy should be actively treated, as it is associated with poor prognosis. The form of MIRAS, starting as acute, treatment resistant epilepsy, is important to diagnose, since valproate therapy almost always leads to acute liver failure requiring liver transplantation.

Infantile-onset spinocerebellar ataxia (IOSCA) is a severe neurodegenerative disorder caused by the recessive mutation in PEO1, leading to an Y508C change in the mitochondrial helicase Twinkle, in its helicase domain. However, no mitochondrial dysfunction has been found in this disease. We studied here the consequences of IOSCA for the central nervous system, as well as the in vitro performance of the IOSCA mutant protein. The results of the mtDNA analyses were compared to findings in a similar juvenile or adult-onset ataxia syndrome, mitochondrial recessive ataxia syndrome (MIRAS), caused by the W748S mutation in the mitochondrial DNA polymerase (POLG). We show here that IOSCA brain does not harbor mtDNA deletions or increased amount of mtDNA point mutations, whereas MIRAS brain shows multiple deletions of mtDNA. However, IOSCA, and to a lesser extent also MIRAS, show mtDNA depletion in the brain and the liver. In both diseases, especially large neurons show respiratory chain complex I (CI) deficiency, but also CIV is decreased in IOSCA. Helicase activity, hexamerization and nucleoid structure of the IOSCA mutant were, however, unaffected. The lack of in vitro helicase defect or cell culture phenotype suggest that Twinkle-Y508C dysfunction affects mtDNA maintenance in a highly context and cell-type specific manner. Our results indicate that IOSCA is a new member of the mitochondrial DNA depletion syndromes.

Twinkle is a mitochondrial replicative helicase, the mutations of which have been associated with autosomal dominant progressive external ophthalmoplegia (adPEO), and recessively inherited infantile onset spinocerebellar ataxia (IOSCA). We report here a new phenotype in two siblings with compound heterozygous Twinkle mutations (A318T and Y508C), characterized by severe early onset encephalopathy and signs of liver involvement. The clinical manifestations included hypotonia, athetosis, sensory neuropathy, ataxia, hearing deficit, ophthalmoplegia, intractable epilepsy and elevation of serum transaminases. The liver showed mtDNA depletion, whereas the muscle mtDNA was only slightly affected. Alpers-Huttenlocher syndrome has previously been associated with mutations of polymerase gamma, a replicative polymerase of mtDNA. We show here that recessive mutations of the close functional partner of the polymerase, the Twinkle helicase, can also manifest as early encephalopathy with liver involvement, a phenotype reminiscent of Alpers syndrome, and are a new genetic cause underlying tissue-specific mtDNA depletion.

Objective: Dysfunction of mitochondrial DNA polymerase gamma (POLG) has been recently recognized as an important cause of inherited neurodegenerative diseases. We have reported dominant and recessive inheritance of parkinsonism, mitochondrial myopathy, and premature amenorrhea in five ethnically distinct families with POLG1 mutations. This prompted us to carry out a detailed analysis of the coding region and intron-exon boundaries of POLG1 in Finnish patients with idiopathic sporadic Parkinson disease (PD) and in nonparkinsonian controls., Methods: The coding region of POLG1 was analyzed in 140 Finnish patients with PD and their 127 spouses as age- and ethnically matched controls. Further, we analyzed the intragenic CAG-repeat region of POLG1 in 126 additional patients with nonparkinsonian neurologic disorders and in 516 Finnish population controls., Results: We found clustering of rare variants of the POLG1 CAG-repeat, encoding a polyglutamine tract, in Finnish patients with idiopathic PD as compared to their spouses (p = 0.003; OR 3.01, 95% CI 1.35 to 6.71), population controls (p = 0.001; OR 2.45, 95% CI 1.45 to 4.14), and patients with nonparkinsonian neurologic disorders (p = 0.05, OR 1.98, 95% CI 0.97 to 4.05). We found several amino acid substitutions, none of them associating with PD. These included a previously parkinsonism-associated POLG variant Y831C, found in one patient with PD, but also in five controls, suggesting that it is a neutral amino acid polymorphism., Conclusions: Our results suggest that POLG polyglutamine tract variants should be considered as a predisposing genetic factor in idiopathic sporadic Parkinson disease.

We reported previously that the DNA polymerase gamma (POLG) W748S mutation, a common cause of mitochondrial recessive ataxia syndrome (MIRAS), has a common ancient founder for all the disease chromosomes in Finland, Norway, United Kingdom, and Belgium. Here, we present results showing that the same ancestral chromosome underlies MIRAS and Alpers syndrome in Australia and New Zealand. Furthermore, we show that a second common POLG mutation, A467T, also shows common European ancestry: patients from Australia, New Zealand, and the United States share a common haplotype with the previously reported European patients. These data of ancestral haplotypes indicate that the POLG locus is quite stable and that the recessive W748S and A467T mutations, and probably also G848S, have occurred once in history. They have effectively spread to populations of European descent with carrier frequencies up to 1% in several populations. Our data predict that these mutations are common causes of ataxia and Alpers disease in the Western world.

Mutations in the catalytic subunit of the mitochondrial DNA polymerase gamma (POLG) have been found to be an important cause of neurological disease. Recently, we and collaborators reported a new neurodegenerative disorder with autosomal recessive ataxia in four patients homozygous for two amino acid changes in POLG: W748S in cis with E1143G. Here, we studied the frequency of this allele and found it to be among the most common genetic causes of inherited ataxia in Finland. We identified 27 patients with mitochondrial recessive ataxia syndrome (MIRAS) from 15 Finnish families, with a carrier frequency in the general population of 1 : 125. Since the mutation pair W748S+E1143G has also been described in European patients, we examined the haplotypes of 13 non-Finnish, European patients with the W748S mutation. Haplotype analysis revealed that all the chromosomes carrying these two changes, in patients from Finland, Norway, the United Kingdom, and Belgium, originate from a common ancient founder. In Finland and Norway, long, common, northern haplotypes, outside the core haplotype, could be identified. Despite having identical homozygous mutations, the Finnish patients with this adult- or juvenile-onset disease had surprisingly heterogeneous phenotypes, albeit with a characteristic set of features, including ataxia, peripheral neuropathy, dysarthria, mild cognitive impairment, involuntary movements, psychiatric symptoms, and epileptic seizures. The high carrier frequency in Finland, the high number of patients in Norway, and the ancient European founder chromosome indicate that this newly identified ataxia should be considered in the first-line differential diagnosis of progressive ataxia syndromes.